Nitric oxide (NO) is a free radical with diverse physiological functions one of which is its smooth muscle relaxant effects. By virtue of this effect NO plays a key role in regulating vascular tone and therefore 1: flow to many organs including the reproductive tract. Recent identification of predominantly endothelial nitric oxide synthese (eNOS) in the human uterus with primary localization to the endometrial glands has raised the possibility that this molecule may have functions other than regulation of blood flow, such as control of endometrial glandular secretion. Additionally, the marked increase in the endometrial expression of eNOS mRNA and protein around the expected time of implantation with a decline just prior menstruation suggests that endometrial eNOS is regulated by sex steroids, and plays a role in implantation process. In this proposal we will test the hypothesis that sex hormones regulate endometrial eNOS, and NO in turn functions as a mediator of estrogenic influence on cellular proliferation, progesterone's effect in induction of endometrial decidualization. To test our hypothesis we will use in vitro approach using primary human derived endometrial cells to test the direct effects of estrogen progesterone and their combination on eNOS expression and NO secretion (Specific Aim 1) Using pharmacological tools to block the synthesis of endometrial NO, and transfection studies to upregulate eNOS gene expression we will determine if NO mediates E and P actions in the endometrium or independent of sex steroids influence cellular proliferation and endometrial secretion of decidual products (Specific Aims 2 and 3). To complement these studies we will use an ex vivo approach to determine if patients with implantation failures may have endometrial eNOS defects Specific Aim 4). This pilot study should shed light on regulation and function of human endometrial NO pathway, an uninvestigated area of research with profound clinical significance.